U.S. patent application number 16/377752 was filed with the patent office on 2019-10-31 for heterocyclic substituted 2-amino quinazoline derivatives for the treatment of viral infections.
The applicant listed for this patent is JANSSEN SCIENCES IRELAND UNLIMITED COMPANY. Invention is credited to Werner Embrechts, Tim Hugo Maria Jonckers, Stefaan Julien Last, David Craig Mc Gowan, Serge Maria Aloysius Pieters, Pierre Jean-Marie Bernard Raboisson.
Application Number | 20190330160 16/377752 |
Document ID | / |
Family ID | 47189782 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330160 |
Kind Code |
A1 |
Last; Stefaan Julien ; et
al. |
October 31, 2019 |
HETEROCYCLIC SUBSTITUTED 2-AMINO QUINAZOLINE DERIVATIVES FOR THE
TREATMENT OF VIRAL INFECTIONS
Abstract
This invention relates to heterocyclic substituted
2-amino-quinazoline derivatives, processes for their preparation,
pharmaceutical compositions, and their use in treating viral
infections.
Inventors: |
Last; Stefaan Julien; (Lint,
BE) ; Mc Gowan; David Craig; (Brussel, BE) ;
Embrechts; Werner; (Beerse, BE) ; Pieters; Serge
Maria Aloysius; (Ar Hulst, NL) ; Jonckers; Tim Hugo
Maria; (Heist-op-den-Berg, BE) ; Raboisson; Pierre
Jean-Marie Bernard; (Wavre, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JANSSEN SCIENCES IRELAND UNLIMITED COMPANY |
Co Cork |
|
IE |
|
|
Family ID: |
47189782 |
Appl. No.: |
16/377752 |
Filed: |
April 8, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15591473 |
May 10, 2017 |
10253003 |
|
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16377752 |
|
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|
14443305 |
May 15, 2015 |
9663474 |
|
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PCT/EP2013/073901 |
Nov 15, 2013 |
|
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15591473 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/12 20180101;
C07D 239/95 20130101; A61P 43/00 20180101; C07D 401/12 20130101;
C07D 417/12 20130101; A61P 37/02 20180101; C07D 239/84 20130101;
C07D 413/12 20130101; A61P 29/00 20180101; C07D 403/12
20130101 |
International
Class: |
C07D 239/84 20060101
C07D239/84; C07D 417/12 20060101 C07D417/12; C07D 413/12 20060101
C07D413/12; C07D 239/95 20060101 C07D239/95; C07D 401/12 20060101
C07D401/12; C07D 403/12 20060101 C07D403/12 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 16, 2012 |
EP |
12192970.7 |
Claims
1. A method of treating a viral infection in which the modulation
of TLR7 and/or TLR 8 is involved in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a compound of formula (I) ##STR00047## or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 is
selected from the group consisting of: ##STR00048## R.sub.2 is
selected from the group consisting of hydrogen,
--O--(C.sub.1-3)-alkyl, halogen, (C.sub.1-3)-alkyl,
--O--(C.sub.1-3)-alkyl-O--(C.sub.1-3)-alkyl and CH.sub.2OH; R.sub.3
is selected from the group consisting of hydrogen,
--O--(C.sub.1-3)-alkyl, halogen, (C.sub.1-3)-alkyl and
--C(.dbd.O)--R.sub.7, wherein R.sub.7 is selected from the group
consisting of --O--(C.sub.1-3)-alkyl, NH.sub.2, NH(CH.sub.3),
N(CH.sub.3).sub.2, N(CH.sub.3)(C.sub.1-3)-alkyl,
N((C.sub.1-3)-alkyl).sub.2 and pyrolidine; R.sub.4 is hydrogen or
fluorine; R.sub.5 is selected from the group consisting of
(C.sub.1-3)-alkyl, (C.sub.1-3)-fluoro-alkyl, and CH.sub.2OH;
R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are each
independently selected from the group consisting of hydrogen,
(C.sub.1-3)-alkyl, --O--(C.sub.1-3)-alkyl and halogen; and R.sub.13
is selected from the group consisting of hydrogen,
(C.sub.1-3)-alkyl and (C.sub.1-3)-fluoro-alkyl.
2-5. (canceled)
6. The method of claim 1, wherein R.sub.5 is CH.sub.3, and R.sub.9
and R.sub.11 are each independently H or CH.sub.3.
7. The method of claim 1, wherein R.sub.13 is H, CH.sub.3 or
CH.sub.2CH.sub.3,
8. The method of claim 1, wherein R.sub.1 is ##STR00049##
9. The method of claim 8, wherein R.sub.9 is CH.sub.3.
10. The method of claim 8, wherein R.sub.2 is OCH.sub.3 or
--O--C.sub.2H.sub.4--O--CH.sub.3.
11. The method of claim 8, wherein: R.sub.3, R.sub.4, R.sub.13 and
R.sub.8 are each H; R.sub.9 is CH.sub.3; and R.sub.2 is
--O--(C.sub.1-3)-alkyl-O--(C.sub.1-3)-alkyl.
12. The method of claim 8, wherein: R.sub.3, R.sub.4, R.sub.13 and
R.sub.8 are each H; R.sub.9 is CH.sub.3; and R.sub.2 is
OCH.sub.3.
13. The method of claim 1, wherein R.sub.1 is ##STR00050##
14. The method of claim 13, wherein R.sub.5 is CH.sub.3; and
R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are each H.
15. The method of claim 1, wherein R.sub.1 is ##STR00051##
16. The method of claim 15, wherein R.sub.5 is CH.sub.3 or
CH.sub.2OH.
17. The method of claim 1, wherein R.sub.1 is ##STR00052##
18. The method of claim 17, wherein: R.sub.5 is CH.sub.3; and
R.sub.8 and R.sub.9 are each H.
19. The method of claim 1, wherein R.sub.1 is ##STR00053##
20. The method of claim 19, wherein each R.sub.5 is CH.sub.3; and
R.sub.8 and R.sub.9 are each H.
21. The method of claim 8, wherein R.sub.1 is ##STR00054##
22. The method of claim 21, wherein each R.sub.13 is
(C.sub.1-3)-alkyl.
23. The method of claim 1, wherein R.sub.1 is ##STR00055##
24. The method of claim 23, wherein each R.sub.13 is
(C.sub.1-3)-alkyl.
25. The method of claim 1, wherein the compound is selected from
the group consisting of: ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060##
26. The method of claim 1, wherein the compound is together with
one or more pharmaceutically acceptable excipients, diluents or
carriers in a pharmaceutical composition.
27. The method of claim 1, wherein the treatment stimulates the
innate immune system of the subject through activation of TLR7
and/or TLR8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/591,473, filed on May 10, 2017, which is a
continuation of U.S. patent application Ser. No. 14/443,305, filed
on May 15, 2015, now U.S. Pat. No. 9,663,474, which is a national
phase entry of International Application No. PCT/EP2013/073901
filed Nov. 15, 2013, which claims priority to European patent
application EP 12192970.7 filed Nov. 16, 2012, each of which are
incorporated herein in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. This ASCII copy, created
on Apr. 4, 2019, is named TIP0289USCNT2_SL.txt and is 657 bytes in
size.
[0003] This invention relates to heterocyclic substituted
2-amino-quinazoline derivatives, processes for their preparation,
pharmaceutical compositions, and their use in treating viral
infections.
[0004] The present invention relates to the use of heterocyclic
substituted 2-amino-quinazoline derivatives in the treatment of
viral infections, immune or inflammatory disorders, whereby the
modulation, or agonism, of toll-like-receptors (TLRs) is involved.
Toll-Like Receptors are primary transmembrane proteins
characterized by an extracellular leucine rich domain and a
cytoplasmic extension that contains a conserved region. The innate
immune system can recognize pathogen-associated molecular patterns
via these TLRs expressed on the cell surface of certain types of
immune cells. Recognition of foreign pathogens activates the
production of cytokines and upregulation of co-stimulatory
molecules on phagocytes. This leads to the modulation of T cell
behaviour.
[0005] It has been estimated that most mammalian species have
between ten and fifteen types of Toll-like receptors. Thirteen TLRs
(named TLR1 to TLR13) have been identified in humans and mice
together, and equivalent forms of many of these have been found in
other mammalian species. However, equivalents of certain TLR found
in humans are not present in all mammals. For example, a gene
coding for a protein analogous to TLR10 in humans is present in
mice, but appears to have been damaged at some point in the past by
a retrovirus. On the other hand, mice express TLRs 11, 12, and 13,
none of which are represented in humans. Other mammals may express
TLRs which are not found in humans. Other non-mammalian species may
have TLRs distinct from mammals, as demonstrated by TLR14, which is
found in the Takifugu pufferfish. This may complicate the process
of using experimental animals as models of human innate
immunity.
[0006] For reviews on TLRs see the following journal articles.
Hoffmann, J. A., Nature, 426, p 33-38, 2003; Akira, S., Takeda, K.,
and Kaisho, T., Annual Rev. Immunology, 21, p 335-376, 2003;
Ulevitch, R. J., Nature Reviews: Immunology, 4, p 512-520,
2004.
[0007] Compounds indicating activity on Toll-Like receptors have
been previously described such as purine derivatives in WO
2006/117670, adenine derivatives in WO 98/01448 and WO 99/28321,
and pyrimidines in WO 2009/067081.
[0008] However, there exists a strong need for novel Toll-Like
receptor modulators having preferred selectivity, higher potency,
higher metabolic stability, and an improved safety profile compared
to the compounds of the prior art.
[0009] In accordance with the present invention a compound of
formula (I) is provided
##STR00001##
or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric
forms, solvate or polymorph thereof, wherein
[0010] R.sub.1 is any of the following structures
##STR00002##
[0011] R.sub.2 is hydrogen, --O--(C.sub.1-3)-alkyl, halogen,
(C.sub.1-3)-alkyl, --O--(C.sub.1-3)-alkyl-O--(C.sub.1-3)-alkyl or
CH.sub.2OH;
[0012] R.sub.3 is hydrogen, --O--(C.sub.1-3)-alkyl, halogen,
(C.sub.1-3)-alkyl or --C(.dbd.O)--R.sub.7 wherein R.sub.7 is
--O--(C.sub.1-3)-alkyl, NH.sub.2, NH(CH.sub.3), N(CH.sub.3).sub.2,
N(CH.sub.3)(C.sub.1-3)-alkyl, N((C.sub.1-3)-alkyl).sub.2 or
pyrolidine;
[0013] R.sub.4 is hydrogen or fluorine;
[0014] R.sub.5 is (C.sub.1-3)-alkyl, (C.sub.1-3)-fluoro-alkyl or
CH.sub.2OH;
[0015] R.sub.6 is NH.sub.2, NH(CH.sub.3) or N(CH.sub.3).sub.2,
(hetero)-anilines optionally substituted with one or more R.sub.8,
R.sub.9, R.sub.10 R.sub.11 or R.sub.12 or (hetero)-benzylamines
optionally substituted with one or more R.sub.8, R.sub.9, R.sub.10
R.sub.11 or R.sub.12,
[0016] R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 which are
the same or different, are each independently selected from
hydrogen, (C.sub.1-3)-alkyl, --O--(C.sub.1-3)-alkyl or halogen
[0017] and
[0018] R.sub.13 is hydrogen, (C.sub.1-3)-alkyl or
(C.sub.1-3)-fluoro-alkyl.
[0019] Preferred compounds according to the invention are compounds
with the numbers 12 and 29 as depicted in Table II.
[0020] The compounds of formula (I) and their pharmaceutically
acceptable salts, tautomer(s), stereo-isomeric forms, solvate or
polymorph thereof have activity as pharmaceuticals, in particular
as modulators of Toll-Like Receptors (especially TLR7 and/or TLR8
activity).
[0021] In a further aspect the present invention provides a
pharmaceutical composition comprising a compound of formula (I) or
a pharmaceutically acceptable salt, tautomer, stereo-isomeric form,
solvate or polymorph thereof together with one or more
pharmaceutically acceptable excipients, diluents or carriers.
[0022] Furthermore a compound of formula (I) or a pharmaceutically
acceptable salt, solvate, tautomer, stereo-isomeric form or
polymorph thereof according to the current invention, or a
pharmaceutical composition comprising said compound of formula(I)
or a pharmaceutically acceptable salt, solvate, tautomer,
stereo-isomeric form or polymorph thereof can be used as a
medicament.
[0023] Another aspect of the invention is that a compound of
formula (I) or its pharmaceutically acceptable salt, solvate,
tautomer, stereo-isomeric form or polymorph thereof, or said
pharmaceutical composition comprising said compound of formula (I)
or a pharmaceutically acceptable salt, solvate, tautomer,
stereo-isomeric form or polymorph thereof can be used accordingly
in the treatment of a disorder in which the modulation of TLR7
and/or TLR8 is involved.
[0024] The term "(C.sub.1-3)-alkyl" refers to a straight-chain,
branched-chain or cyclic saturated aliphatic hydrocarbon containing
the specified number of carbon atoms.
[0025] The term "(C.sub.1-3)-fluoro-alkyl" refers to a
straight-chain, branched-chain or cyclic saturated aliphatic
hydrocarbon containing the specified number of carbon atoms where
one or more hydrogen atoms was replaced by a fluorine atom.
[0026] The term "halogen" refers to fluorine, chlorine, bromine or
iodine, preferably to fluorine and chlorine.
[0027] The term "aniline" refers a compound with the formula
C.sub.6H.sub.5NR.sub.13-- consisting of a phenyl group attached to
an amino group; with "(hetero)-aniline" is meant that in the
aromatic ring 1-3 nitrogen atoms, preferably 1 nitrogen atom, are
present.
[0028] The term "benzylamine" means a compound of the formula
C.sub.6H.sub.5CH.sub.2NR.sub.13-- consisting of a benzyl group,
C.sub.6H.sub.5CH.sub.2, attached to an amine functional group; with
"(hetero)-benzylamine" is meant that in the aromatic ring 1-3
nitrogen atoms, preferably 1 nitrogen atom, are present.
[0029] As used herein, any chemical formula with bonds shown only
as solid lines and not as solid wedged or hashed wedged bonds, or
otherwise indicated as having a particular configuration (e.g. R,
S) around one or more atoms, contemplates each possible
stereoisomer, or mixture of two or more stereoisomers.
[0030] The terms "stereoisomers", "stereoisomeric forms" or
"stereochemically isomeric forms" hereinbefore or hereinafter are
used interchangeably.
[0031] The invention includes all stereoisomers of the compounds of
the invention either as a pure stereoisomer or as a mixture of two
or more stereoisomers.
[0032] Enantiomers are stereoisomers that are non-superimposable
mirror images of each other. A 1:1 mixture of a pair of enantiomers
is a racemate or racemic mixture.
[0033] Diastereomers (or diastereoisomers) are stereoisomers that
are not enantiomers, i.e. they are not related as mirror images. If
a compound contains a double bond, the substituents may be in the E
or the Z configuration. If a compound contains an at least
disubstituted non-aromatic cyclic group, the substituents may be in
the cis or trans configuration.
[0034] Therefore, the invention includes enantiomers,
diastereomers, racemates, E isomers, Z isomers, cis isomers, trans
isomers and mixtures thereof, whenever chemically possible.
[0035] The meaning of all those terms, i.e. enantiomers,
diastereomers, racemates, E isomers, Z isomers, cis isomers, trans
isomers and mixtures thereof are known to the skilled person.
[0036] The absolute configuration is specified according to the
Cahn-lngold-Prelog system. The configuration at an asymmetric atom
is specified by either R or S.
[0037] Resolved stereoisomers whose absolute configuration is not
known can be designated by (+) or (-) depending on the direction in
which they rotate plane polarized light. For instance, resolved
enantiomers whose absolute configuration is not known can be
designated by (+) or (-) depending on the direction in which they
rotate plane polarized light.
[0038] When a specific stereoisomer is identified, this means that
said stereoisomer is substantially free, i.e. associated with less
than 50%, preferably less than 20%, more preferably less than 10%,
even more preferably less than 5%, in particular less than 2% and
most preferably less than 1%, of the other stereoisomers. Thus,
when a compound of Formula (I) is for instance specified as (R),
this means that the compound is substantially free of the (S)
isomer; when a compound of Formula (I) is for instance specified as
E, this means that the compound is substantially free of the Z
isomer; when a compound of Formula (I) is for instance specified as
cis, this means that the compound is substantially free of the
trans isomer.
[0039] Pharmaceutically acceptable salts of the compounds of
formula (I) include the acid addition and base salts thereof.
Suitable acid addition salts are formed from acids which form
non-toxic salts. Suitable base salts are formed from bases which
form non-toxic salts.
[0040] The compounds of the invention may also exist in unsolvated
and solvated forms. The term "solvate" is used herein to describe a
molecular complex comprising the compound of the invention and one
or more pharmaceutically acceptable solvent molecules, for example,
ethanol.
[0041] The term "polymorph" refers to the ability of the compound
of the invention to exist in more than one form or crystal
structure.
[0042] The compounds of the present invention may be administered
as crystalline or amorphous products. They may be obtained for
example as solid plugs, powders, or films by methods such as
precipitation, crystallization, freeze drying, spray drying, or
evaporative drying. They may be administered alone or in
combination with one or more other compounds of the invention or in
combination with one or more other drugs. Generally, they will be
administered as a formulation in association with one or more
pharmaceutically acceptable excipients. The term "excipient" is
used herein to describe any ingredient other than the compound(s)
of the invention. The choice of excipient depends largely on
factors such as the particular mode of administration, the effect
of the excipient on solubility and stability, and the nature of the
dosage form.
[0043] The compounds of the present invention or any subgroup
thereof may be formulated into various pharmaceutical forms for
administration purposes. As appropriate compositions there may be
cited all compositions usually employed for systemically
administering drugs. To prepare the pharmaceutical compositions of
this invention, an effective amount of the particular compound,
optionally in addition salt form, as the active ingredient is
combined in intimate admixture with a pharmaceutically acceptable
carrier, which carrier may take a wide variety of forms depending
on the form of preparation desired for administration. These
pharmaceutical compositions are desirably in unitary dosage form
suitable, for example, for oral, rectal, or percutaneous
administration. For example, in preparing the compositions in oral
dosage form, any of the usual pharmaceutical media may be employed
such as, for example, water, glycols, oils, alcohols and the like
in the case of oral liquid preparations such as suspensions,
syrups, elixirs, emulsions, and solutions; or solid carriers such
as starches, sugars, kaolin, diluents, lubricants, binders,
disintegrating agents and the like in the case of powders, pills,
capsules, and tablets. Because of their ease in administration,
tablets and capsules represent the most advantageous oral dosage
unit forms, in which case solid pharmaceutical carriers are
obviously employed. Also included are solid form preparations that
can be converted, shortly before use, to liquid forms. In the
compositions suitable for percutaneous administration, the carrier
optionally comprises a penetration enhancing agent and/or a
suitable wetting agent, optionally combined with suitable additives
of any nature in minor proportions, which additives do not
introduce a significant deleterious effect on the skin. Said
additives may facilitate the administration to the skin and/or may
be helpful for preparing the desired compositions. These
compositions may be administered in various ways, e.g., as a
transdermal patch, as a spot-on, as an ointment. The compounds of
the present invention may also be administered via inhalation or
insufflation by means of methods and formulations employed in the
art for administration via this way. Thus, in general the compounds
of the present invention may be administered to the lungs in the
form of a solution, a suspension or a dry powder.
[0044] It is especially advantageous to formulate the
aforementioned pharmaceutical compositions in unit dosage form for
ease of administration and uniformity of dosage. Unit dosage form
as used herein refers to physically discrete units suitable as
unitary dosages, each unit containing a predetermined quantity of
active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such unit dosage forms are tablets (including scored or
coated tablets), capsules, pills, powder packets, wafers,
suppositories, injectable solutions or suspensions and the like,
and segregated multiples thereof.
[0045] Those of skill in the treatment of infectious diseases will
be able to determine the effective amount from the test results
presented hereinafter. In general it is contemplated that an
effective daily amount would be from 0.01 mg/kg to 50 mg/kg body
weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It
may be appropriate to administer the required dose as two, three,
four or more sub-doses at appropriate intervals throughout the day.
Said sub-doses may be formulated as unit dosage forms, for example,
containing 1 to 1000 mg, and in particular 5 to 200 mg of active
ingredient per unit dosage form.
[0046] The exact dosage and frequency of administration depends on
the particular compound of formula (I) used, the particular
condition being treated, the severity of the condition being
treated, the age, weight and general physical condition of the
particular patient as well as other medication the individual may
be taking, as is well known to those skilled in the art.
Furthermore, it is evident that the effective amount may be lowered
or increased depending on the response of the treated subject
and/or depending on the evaluation of the physician prescribing the
compounds of the instant invention. The effective amount ranges
mentioned above are therefore only guidelines and are not intended
to limit the scope or use of the invention to any extent.
Preparation of Compounds of Formula (I)
[0047] Compounds of formula (I) are prepared according to scheme 1.
Substituted anthranilic esters or acids (II) were heated under
acidic conditions in the presence of excess cyanamide, using an
alcoholic solvent (e.g. ethanol) or diglyme according to the method
described in the literature (O'Hara et. al. JOC (1991) 56, p 776).
Subsequent amine substitution of the 2-amino-4-hydroxyquinazolines
(III) can proceed via a coupling agent such as BOP or PyBOP in the
presence of DBU and the amine in a polar aprotic solvent (e.g.
DMF).
##STR00003##
EXPERIMENTAL SECTION
General Procedure of Making a Substituted
2-amino-4-hydroxyquinazoline
##STR00004##
[0049] Into a 500 mL pressure vessel equipped with a magnetic stir
bar was placed 2-amino-6-methoxybenzoic acid (25 g, 149.6 mmol),
ethanol (200 mL), cyanamide (9.43 g, 224 mmol), and concentrated
HCl (6 mL). The mixture was allowed to stir at 100.degree. C. for
16 h. The reaction mixture was allowed to cool to room temperature
and the solids were isolated via filtration and washed with ethanol
and DIPE. The crude product was dried under vacuum at 50.degree. C.
to obtain an off white solid.
[0050] LC-MS m/z=192(M+H)
[0051] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 3.88 (s,
3H), 6.96 (dd, J=8.2, 3.1 Hz, 2H), 7.69 (t, J=8.3 Hz, 1H), 8.28
(br. s., 2H), 12.67 (br. s., 1H)
TABLE-US-00001 TABLE I Compounds of formula (III). The following
intermediates were prepared according to the method to prepare
III-1. LCMS # STRUCTURE H NMR (M+H).sup.+ 1 ##STR00005## .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 6.98 (dd, J = 11.0, 8.3 Hz,
1 H), 7.13 (d, J = 8.3 Hz, 1 H), 7.51 (br. s., 2 H), 7.64 (td, J =
8.3, 5.8 Hz, 1 H), 12.30 (br. s, 1 H) 180 2 ##STR00006## .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 7.01-7.16 (m, 2 H), 7.56
(br. s., 2 H) 7.99 (t, J = 7.7 Hz, 1 H), 10.38-13.48 (m, 1 H) 180 3
##STR00007## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
6.51-6.67 (m, 2 H), 7.00-7.08(m, 1 H), 7.42(ddd, J = 11.2, 7.9 1.3
Hz, 1 H), 7.69 (dd, J = 7.9, 0.6 Hz, 1 H), 11.08 (br. s., 1 H) 180
4 ##STR00008## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 2.43
(s, 3 H), 7.22 (d, J = 1.0 Hz, 1 H), 7.24 (s, 1 H), 7.89 (d, J =
8.0 Hz, 1 H), 8.29 (br. s., 2 H), 12.65 (br. s, 1 H) 176 5
##STR00009## Not available 192 6 ##STR00010## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.41 (dd, J = 8.5, 2.0 Hz, 1 H), 7.55 (d,
J = 2.0 Hz, 1 H), 7.98 (d, J = 8.5 Hz, 1 H), 8.49 (br. s., 2 H),
10.79-13.69 (m, 1 H) 196 7 ##STR00011## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 3.87-3.95 (m, 3 H), 7.12-7.47 (m, 1 H),
7.83 (dd, J = 8.3, 1.4 Hz, 1 H), 7.99 (d, J = 1.3 Hz, 1 H), 8.07-
8.13 (m, 1 H), 8.43 (br. s., 2 H) 220 8 ##STR00012## Not available
174 (M-H).sup.- 9 ##STR00013## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 3.74-3.82 (m, 3 H), 6.42 (br. s., 2 H), 6.62 (d, J =
7.7 Hz, 1 H), 6.75 (dd, J = 8.3, 0.8 Hz, 1 H), 7.44 (t, J = 8.3 Hz,
1 H), 10.91 (br. s., 1 H) 192 10 ##STR00014## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.40 (dd, J = 8.7, 4.7 Hz, 1 H), 7.48 (t,
J = 8.8 Hz, 1 H) NA
General Procedure of Making Compound IV
[0052] Compound III (1.5 mmol) and DBU (3.75 mmol) were dissolved
in 5 mL DMF in a 30 mL glass vial. After 5 minutes BOP (1.5 mmol)
was added. The reaction mixture was stirred for 5 minutes and then
the amine (2.25 mmol) was added. The reaction mixture was stirred
overnight. The crude reaction mixture was purified by prep. HPLC on
(RP Vydac Denali C18--10 .mu.m, 250 g, 5 cm). Mobile phase (0.25%
NH.sub.4HCO.sub.3 solution in water, MeOH), the desired fractions
were collected, evaporated, dissolved in MeOH and evaporated again
to obtain the product as a solid.
General Procedure to Make Compounds 22, 23, 24, 26, 27 and 28
[0053] Compound 8 of formula (I) (see table II) (2.1 g, 6.5 mmol)
was dispensed in THF (50 mL), LiOH (409 mg, 9.74 mmol) was added
followed by MeOH (5 mL). The reaction mixture was stirred overnight
at room temperature. The solvents were evaporated until only water
remained. 10 mL 1M HCl was added and the compound was extracted
with 2-methyltetrahydrofuran (2.times.25 mL). The combined organic
layers were dried on MgSO.sub.4 and the solvents were removed under
reduced pressure to obtain
2-amino-4-[1-(2-pyridyl)ethylamino]quinazoline-7-carboxylic acid as
a white solid.
[0054] 2-amino-4-[1-(2-pyridyl)ethylamino]quinazoline-7-carboxylic
acid (200 mg, 0.65 mmol) and PyBOP (421 mg, 0.81 mmol) were
dissolved in DMF (5 mL) in a 30 mL glass vial. After 5 minutes
Hunig's base (0.557 mL, 3.23 mmol) was added. The reaction mixture
was stirred for 5 minutes and then the amine was added. The
reaction mixture was stirred overnight. The crude reaction mixture
was purified by preparative. HPLC on (RP Vydac Denali C18--10
.mu.m, 250 g, 5 cm). Mobile phase (0.25% NH.sub.4HCO.sub.3 solution
in water, MeOH), the desired fractions were collected, evaporated,
dissolved in MeOH and evaporated again to obtain the product as a
solid.
Procedure to Make Compound 29
[0055] Compound 12 of formula (I) (see table II) (1500 mg, 4.78
mmol) and pyridine hydrochloride (3.32 g, 28.7 mmol) were dissolved
in pyridine (20 mL) and heated to 120.degree. C. for 16 h. Pyridine
was removed under reduced pressure. The residual fraction was
quenched with a NaHCO.sub.3(sat., aq.) solution. The precipitate
was filtered off, washed with water and dried under vacuum at
50.degree. C. to afford a brown solid which was purified by
preparative HPLC (Stationary phase: RP Vydac Denali C18--10 .mu.m,
200 g, 5 cm), Mobile phase: 0.25% NH.sub.4HCO.sub.3 solution in
water, CH.sub.3CN), the desired fractions were collected,
evaporated, dissolved in MeOH and evaporated again to obtain
2-amino-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5-ol (100
mg) as a solid.
[0056]
2-amino-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5-ol (40
mg, 0.15 mmol) and Cs.sub.2CO.sub.3 (144 mg, 0.44 mmol) were
dissolved in DMF (7.5 mL) and stirred at room temperature for 30
minutes. 2-bromoethyl methyl ether (0.018 mL, 0.18 mmol) was added
and the entire mixture was stirred for 16 hours at room
temperature. The solvent was removed under reduced pressure and the
crude residue was neutralized with 1M HCl and purified by
preparative HPLC on (RP Vydac Denali C18--10 .mu.m, 250 g, 5 cm).
Mobile phase (0.25% NH.sub.4HCO.sub.3 solution in water, MeOH), the
desired fractions were collected, evaporated, dissolved in MeOH and
evaporated again to obtain compound 29 as a solid.
Procedure to Make Compound 30
[0057] A 75-mL stainless steel autoclave was charged under N.sub.2
atmosphere with 2-amino-5-bromo-quinazolin-4-ol (3 g, 12.5 mmol),
Pd(OAc).sub.2 (56 mg, 0.25 mmol), 1,3 bis(diphenylphosphino)propane
(206 mg, 0.5 mmol), potassium acetate (2.45 g, 25 mmol), methanol
(25 mL) and THF (30 mL). The autoclave was closed and pressurized
to 50 bar CO gas and the reaction was carried out for 16 hours at
100.degree. C. The formed precipitate was removed by filtration
yielding methyl 2-amino-4-hydroxy-quinazoline-5-carboxylate (2.35
g).
[0058] Methyl 2-amino-4-hydroxy-quinazoline-5-carboxylate (2.35 g)
in THF (10 mL) was cooled to 0.degree. C. Then LiAlH.sub.4 was
added. The mixture was allowed to reach room temperature and
stirred for 16 hours. EtOAc (5 mL) was added drop wise at 0.degree.
C., then 3 g Na.sub.2SO.sub.4.10H.sub.2O was added and the entire
mixture was stirred for 30 minutes. The precipitate was filtered
off, and the filtrate was dried with MgSO.sub.4, filtered and
evaporated to dryness to obtain
2-amino-5-(hydroxymethyl)quinazolin-4-ol (750 mg) as a yellow
solid.
[0059] 2-amino-5-(hydroxymethyl)quinazolin-4-ol (300 mg, 1.57 mmol)
was suspended in THF (20 mL) with DBU (0.586 mL, 3.92 mmol), after
5 minutes BOP (833 mg, 1.88 mmol) was added. After 15 minutes
(5-methyl-3-isoxazolyl)methylamine (0.320 mL, 3.14 mmol) was added.
The mixture was stirred for 16 hours at room temperature. The
solvent was removed under reduced pressure and the crude product
was purified by preparative HPLC on (RP Vydac Denali C18--10 .mu.m,
250 g, 5 cm). Mobile phase (0.25% NH.sub.4HCO.sub.3 solution in
water, MeOH), the desired fractions were collected, evaporated,
dissolved in MeOH and evaporated again to obtain compound 30 as a
solid (119 mg).
Procedure to Make Compound 31
[0060] A freshly prepared NaOMe solution (1.25 mL, 6.25 mmol) was
added under N.sub.2 atmosphere to a mixture of
2-amino-5-bromo-8-fluoro-quinazolin-4-ol (500 mg, 1.94 mmol),
copper (I) bromide (39 mg, 0.27 mmol), EtOAc (0.076 mL, 0.78 mmol)
in MeOH (5 mL). The mixture was heated up in a pressure vessel to
reflux for 16 hours. The solvent was removed under reduced
pressure. The residue was purified by preparative HPLC (Stationary
phase: RP Vydac Denali C18--10 .mu.m, 200 g, 5 cm), Mobile phase:
0.25% NH.sub.4HCO.sub.3 solution in water, MeOH), the desired
fractions were collected, evaporated, dissolved in MeOH and
evaporated again to obtain
2-amino-8-fluoro-5-methoxy-quinazolin-4-ol (150 mg) as a solid.
[0061] 2-amino-8-fluoro-5-methoxy-quinazolin-4-ol (150 mg, 0.72
mmol) was dispensed in DMF (10 mL), DBU (0.536 mL, 3.59 mmol), was
added and then BOP reagent (396 mg, 0.90 mmol) was added. The
reaction mixture was stirred and when it was homogeneous
(5-methyl-3-isoxazolyl)methylamine (0.115 mL, 1.08 mmol) was added.
The reaction mixture was stirred 16 hours. The reaction was
concentrated under reduced pressure and the residue was purified by
preparative HPLC (Stationary phase: RP Vydac Denali C18--10 .mu.m,
200 g, 5 cm), Mobile phase: 0.25% NH.sub.4HCO.sub.3 solution in
water, MeOH), the desired fractions were collected, evaporated,
dissolved in MeOH and evaporated again to obtain compound 31 as a
solid (64 mg).
Procedure to Make Compound 32
[0062] Compound 31 (52.5 mg, 0.173 mmol) and pyridine hydrochloride
(0.12 g, 1.039 mmol) in 1 mL pyridine was heated to 120.degree. C.
for 16 hours. The volatiles were removed under reduced pressure.
The residue was quenched with a NaHCO.sub.3 (sat., aq.) solution.
The precipitate was filtered off, washed with water and dried under
vacuum at 50.degree. C. to afford
2-amino-8-fluoro-4-[(5-methylisoxazol-3-yl)methylamino]quinazol-
in-5-ol (10 mg) as a brown solid.
[0063]
2-amino-8-fluoro-4-[(5-methylisoxazol-3-yl)methylamino]quinazolin-5-
-ol (10 mg, 0.035 mmol) and Cs.sub.2CO.sub.3 (33.8 mg, 0.104 mmol)
in DMF (5 mL) was stirred at room temperature for 30 minutes.
2-chloroethyl methyl ether (4.1 mg, 0.043 mmol) was added and the
entire mixture was stirred for 16 hours at room temperature. The
solvent was removed under reduced pressure. The residue was
dissolved in MeOH and the precipitate (salts) were removed by
filtration. The filtrate was concentrated under reduced pressure
and the crude residue was purified by preparative HPLC on
(Stationary phase: RP SunFire Prep C18 OBD-10 .mu.m, 30.times.150
mm), Mobile phase: 0.25% NH.sub.4HCO.sub.3 solution in water,
CH.sub.3CN), the desired fractions were collected, evaporated,
dissolved in MeOH and evaporated again to obtain compound 32 as a
solid (2 mg).
TABLE-US-00002 TABLE II Compounds of formula (I). The following
compounds were synthesized according to one of the methods
described above. # STRUCTURE H NMR 1 ##STR00015## .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 1.60 (d, J = 7.3 Hz, 3 H), 5.61
(quin, J = 7.3 Hz, 1 H), 5.97 (s, 2 H), 7.05 (ddd, J = 8.1, 6.9,
1.2 Hz, 1 H), 7.20 (dd, J = 8.4, 0.7 Hz, 1 H), 7.24 (ddd, J = 7.5,
4.8, 0.9 Hz, 1 H), 7.44 (d, J = 7.9 Hz, 1 H), 7.49 (ddd, J = 8.3,
6.9, 1.3 Hz, 1 H), 7.72 (td, J = 7.7, 1.8 Hz, 1 H), 8.05 (d, J =
7.9 Hz, 1 H), 8.18 (dd, J = 8.3, 1.0 Hz, 1 H), 8.50-8.56 (m, 1 H) 2
##STR00016## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.53
(d, J = 6.82 Hz, 3 H) 3.99 (s, 3 H) 5.43 (t, J = 6.82 Hz, 1 H) 6.03
(s, 2 H) 6.53-6.69 (m, 1 H) 6.81 (dd, J = 8.36, 0.88 Hz, 1 H) 7.32
(ddd, J = 7.48, 4.84, 1.10 Hz, 1 H) 7.38 (t, J = 8.14 Hz, 1 H) 7.46
(d, J = 7.92 Hz, 1 H) 7.80 (td, J = 7.70, 1.76 Hz, 1 H) 8.54-8.72
(m, 1 H) 9.01 (d, J = 7.04 Hz, 1 H) 3 ##STR00017## .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta..quadrature. ppm 4.05 (s, 3 H), 6.25 (s,
2 H), 6.43 (quin, J = 7.8 Hz, 1 H), 6.62-6.68 (m, 1 H), 6.86 (dd, J
= 8.4, 0.9 Hz, 1 H), 7.44 (t, J = 8.1 Hz, 1 H), 7.52 (ddd, J = 7.7,
4.8, 1.1 Hz, 1 H), 7.69 (d, J = 7.7 Hz, 1 H), 7.95 (td, J = 7.7,
1.8 Hz, 1 H), 8.74- 8.79 (m, 1 H), 9.31 (d, J = 8.4 Hz, 1 H) 4
##STR00018## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.60
(d, J = 6.6 Hz, 3 H), 5.34 (br. s., 2 H), 5.49 (t, J = 6.8 Hz, 1
H), 6.78 (td, J = 8.6, 2.6 Hz, 1 H), 7.02 (dd, J = 10.8, 2.6 Hz, 1
H), 7.19 (ddd, J = 7.5, 4.8, 1.1 Hz, 1 H), 7.26-7.31 (m, 1 H), 7.59
(d, J = 6.8 Hz, 1 H), 7.65 (td, J = 7.6, 1.9 Hz, 1 H), 7.73 (dd, J
= 9.0, 5.9 Hz, 1 H), 8.53-8.61 (m, 1 H) 5 ##STR00019## .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 1.58 (d, J = 7.0 Hz, 3 H), 2.35
(s, 3 H), 5.59 (quin, J = 7.3 Hz, 1 H), 5.94 (s, 2 H), 6.90 (dd, J
= 8.3, 1.2 Hz, 1 H), 7.01 (s, 1 6 H), 7.23 (dd, J = 6.9, 5.2 Hz, 1
H), 7.43 (d, J = 7.9 Hz, 1 H), 7.72 (td, J = 7.7, 1.8 Hz, 1 H),
7.97 (d, J = 7.9 Hz, 1 H), 8.07 (d, J = 8.4 Hz, 1 H), 8.48-8.57 (m,
1 H) 6 ##STR00020## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.57 (d, J = 7.04 Hz, 3 H) 3.80 (s, 3 H) 5.58 (t, J = 7.37 Hz, 1 H)
5.89 (s, 2 H) 6.61 (d, J = 2.42 Hz, 1 H) 6.67 (dd, J = 8.91, 2.53
Hz, 1 H) 7.23 (ddd, J = 7.48, 4.84, 0.88 Hz, 1 H) 7.42 (d, J = 7.92
Hz, 1 H) 7.72 (td, J = 7.70, 1.76 Hz, 1 H) 7.89 (d, J = 8.14 Hz, 1
H) 8.08 (d, J = 9.02 Hz, 1 H) 8.52 (dt, J = 3.96, 0.88 Hz, 1 H) 7
##STR00021## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.59
(d, J = 7.3 Hz, 3 H), 5.53-5.65 (m, 1 H), 6.21 (br. s., 2 H), 7.07
(dd, J = 8.7, 2.1 Hz, 1 H), 7.18 (d, J = 2.0 Hz, 1 H), 7.24 (ddd, J
= 7.4, 4.8, 1.0 Hz, 1 H), 7.43 (d, J = 7.9 Hz, 1 H), 7.73 (td, J =
7.6, 1.9 Hz, 1 H), 8.19 (d, J = 7.9 Hz, 1 H), 8.23 (d, J = 8.8 Hz,
1 H), 8.50-8.56 (m, 1 H) 8 ##STR00022## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.60 (d, J = 7.0 Hz, 3 H), 3.88 (s, 3 H),
5.61 (quin, J = 7.2 Hz, 1 H), 6.22 (s, 2 H), 7.25 (ddd, J = 7.5,
4.8, 0.9 Hz, 1 H), 7.45 (d, J = 7.9 Hz, 1 H), 7.54 (dd, J = 8.6,
1.8 Hz, 1 H), 7.70-7.77 (m, 2 H), 8.28 (d, J = 7.9 Hz, 1 H), 8.32
(d, J = 8.6 Hz, 1 H), 8.51-8.57 (m, 1 H) 9 ##STR00023## .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 1.60 (d, J = 7.04 Hz, 3 H) 5.61
(quin, J = 7.26 Hz, 1 H) 6.25 (br. s., 2 H) 6.99 (td, J = 7.98,
4.95 Hz, 1 H) 7.25 (ddd, J = 7.48, 4.84, 0.88 Hz, 1 H) 7.29-7.36
(m, 1 H) 7.44 (d, J = 7.92 Hz, 1 H) 7.73 (td, J = 7.65, 1.87 Hz, 1
H) 8.01 (d, J = 8.14 Hz, 1 H) 8.17 (d, J = 8.14 Hz, 1 H) 8.52- 8.59
(m, 1 H) 10 ##STR00024## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 1.69 (d, J = 7.0 Hz, 3 H), 3.96 (s, 3 H), 5.80 (quin, J
= 7.1 Hz, 1 H), 6.09 (s, 2 H), 6.60 (dd, J = 8.0, 0.8 Hz, 1 H),
6.83 (dd, J = 8.4, 0.9 Hz, 1 H), 7.40 (t, J = 8.3 Hz, 1 H), 7.61
(d, J = 3.1 Hz, 1 H), 7.77 (d, J = 3.3 Hz, 1 H), 8.37 (d, J = 7.7
Hz, 1 H) 11 ##STR00025## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 2.35 (d, J = 0.9 Hz, 3 H), 4.72 (d, J = 5.3 Hz, 2 H),
6.22 (d, J = 0.7 Hz, 1 H), 6.35 (s, 2 H), 6.80 (ddd, J = 12.3, 7.9,
0.9 Hz, 1 H), 7.04 (dd, J = 8.4, 0.9 Hz, 1 H), 7.46 (td, J = 8.2,
6.5 Hz, 1 H), 7.71-7.82 (m, 1 H) 12 ##STR00026## .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 2.36 (d, J = 0.7 Hz, 3 H), 3.92 (s,
3 H), 4.70 (d, J = 5.7 Hz, 2 H), 6.05 (s, 2 H), 6.20 (d, J = 0.7
Hz, 1 H), 6.56 (dd, J = 8.0, 0.8 Hz, 1 H), 6.81 (dd, J = 8.4, 0.9
Hz, 1 H), 7.38 (t, J = 8.1 Hz, 1 H), 8.40 (t, J = 5.8 Hz, 1 H) 13
##STR00027## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
.quadrature. ppm 2.33-2.38 (m, 3 H), 4.67 (d, J = 5.9 Hz, 2 H),
6.18-6.24 (m, 1 H), 6.27 (s, 2 H), 6.85-6.92 (m, 2 H), 7.99-8.07
(m, 1 H), 8.42 (t, J = 5.7 Hz, 1 H) 14 ##STR00028## .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 2.35 (d, J = 0.9 Hz, 3 H), 4.69
(d, J = 5.9 Hz, 2 H), 6.22 (d, J = 0.9 Hz, 1 H), 6.39 (br. s., 2
H), 6.98 (td, J = 8.0, 4.8 Hz, 1 H), 7.33 (ddd, J = 11.4, 7.8, 1.1
Hz, 1 H), 7.79 (d, J = 8.4 Hz, 1 H), 8.48 (t, J = 5.8 Hz, 1 H) 15
##STR00029## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta..quadrature. ppm 0.97 (d, J = 6.6 Hz, 3 H), 3.92 (s, 3 H),
4.44-4.55 (m, 1 H), 4.89 (d, J = 3.1 Hz, 1 H), 5.69 (br. s., 1 H),
6.06 (s, 2 H), 6.52-6.58 (m, 1 H), 6.79 (dd, J = 8.3, 0.8 Hz, 1 H),
7.22-7.29 (m, 1 H), 7.32-7.41 (m, 3 H), 7.43-7.49 (m, 2 H), 8.07
(d, J = 7.9 Hz, 1 H) 16 ##STR00030## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.51 (d, J = 6.8 Hz, 3 H), 3.79 (s, 3 H),
3.90 (s, 3 H), 5.39 (quin, J = 7.0 Hz, 1 H), 6.05 (s, 2 H),
6.52-6.58 (m, 1 H), 6.79 (dd, J = 8.4, 0.9 Hz, 1 H), 7.35 (t, J =
8.3 Hz, 1 H), 7.45 (s, 1 H), 7.68 (s, 1 H), 7.84 (d, J = 7.7 Hz, 1
H) 17 ##STR00031## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
3.40- 3.49 (m, 1 H), 3.60 - 3.71 (m, 1 H), 4.45-4.55 (m, 1 H), 4.79
(br. s., 1 H), 4.97-5.05 (m, 1 H), 5.62 (d, J = 4.8 6 Hz, 1 H),
5.98 (s, 2 H), 7.02 (t, J = 7.5 Hz, 1 H), 7.08 (d, J = 8.3 Hz, 1
H), 7.13-7.21 (m, 2 H), 7.27 (t, J = 7.5 Hz, 2 H), 7.38 (d, J = 7.3
Hz, 2 H), 7.42-7.50 (m, 1 H), 7.95 (d, J = 8.3 Hz, 1 H) 18
##STR00032## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.55
(d, J = 6.82 Hz, 3 H) 5.49 (td, J = 6.77, 2.09 Hz, 1 H) 6.32 (s, 2
H) 6.82 (ddd, J = 12.76, 7.92, 0.88 Hz, 1 H) 7.05 (dd, J = 8.47,
0.99 Hz, 1 H) 7.33 (ddd, J = 7.54, 4.90, 0.99 Hz, 1 H) 7.42-7.57
(m, 2 H) 7.82 (td, J = 7.70, 1.76 Hz, 1 H) 7.93 (dd, J = 14.63,
6.93 Hz, 1 H) 8.58-8.67 (m, 1 H) 19 ##STR00033## .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 2.33- 2.39 (m, 3 H), 2.75 (s, 3 H),
4.71 (d, J = 5.3 Hz, 2 H), 6.06 (s, 2 H), 6.22-6.26 (m, 1 H), 6.82
(d, J = 6.8 Hz, 1 H), 7.08 (d, J = 7.7 Hz, 1 H), 7.13 (t, J = 5.3
Hz, 1 H), 7.32 (dd, J = 8.4, 7.3 Hz, 1 H) 20 ##STR00034## .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.49 (d, J = 6.6 Hz, 3 H),
2.58 (s, 3 H), 4.02 (s, 3 H), 5.37 (quin, J = 6.6 Hz, 1 H), 6.02
(s, 2 H), 6.56- 6.62 (m, 1 H), 6.81 6 (dd, J = 8.3, 0.8 Hz, 1 H),
7.20 (d, J = 7.5 Hz, 1 H), 7.24 (d, J = 7.7 Hz, 1 H), 7.37 (t, J =
8.1 Hz, 1 H), 7.70 (t, J = 7.7 Hz, 1 H), 9.21 (d, J = 6.8 Hz, 1 H)
21 ##STR00035## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.36 (t, J = 7.3 Hz, 3 H), 1.57 (d, J = 6.8 Hz, 3 H), 3.95 (s, 3
H), 4.21- 4.42 (m, 2 H), 5.65 (quin, J = 7.0 Hz, 1 H), 6.08 (br.
s., 2 H), 6.58 (dd, J = 7.9, 0.7 Hz, 1 H), 6.81 (dd, J = 8.4, 0.7
Hz, 1 H), 7.39 (t, J = 8.1 Hz, 1 H), 7.88 (s, 1 H), 8.26 (d, J =
7.7 Hz, 1 H) 22 ##STR00036## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 1.60 (d, J = 7.3 Hz, 3 H), 2.80 (d, J = 4.4 Hz, 3 H),
5.60 (quin, J = 7.3 Hz, 1 H), 6.11 (s, 2 H), 7.24 (ddd, J = 7.4,
4.8, 1.0 Hz, 1 H), 7.45 (dt, J = 8.4, 1.8 Hz, 2 H), 7.65 (d, J =
1.8 Hz, 1 H), 7.73 (td, J = 7.7, 1.8 Hz, 1 H), 8.16 (d, J = 7.9 Hz,
1 H), 8.25 (d, J = 8.4 Hz, 1 H), 8.49-8.56 (m, 2 H) 23 ##STR00037##
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.60 (d, J = 7.0
Hz, 3 H), 2.91 (s, 3 H), 3.00 (s, 3 H), 5.61 (quin, J = 7.3 Hz, 1
H), 6.12 (s, 2 H), 7.02 (dd, J = 8.3, 1.7 Hz, 1 6 H), 7.12 (d, J =
1.5 Hz, 1 H), 7.24 (ddd, J = 7.5, 4.8, 0.9 Hz, 1 H), 7.44 (d, J =
7.9 Hz, 1 H), 7.73 (td, J = 7.7, 1.8 Hz, 1 H), 8.16 (d, J = 7.9 Hz,
1 H), 8.24 (d, J = 8.4 Hz, 1 H), 8.51-8.56 (m, 1 H) 24 ##STR00038##
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.03-1.20 (m, 6 H),
1.60 (d, J = 7.0 Hz, 3 H), 2.68-2.89 (m, 3 H), 3.76-3.91 (m, 1 H),
5.61 (quin, J = 7.2 Hz, 1 H), 6.13 (br. s., 2 H), 6.94-7.02 (m, 1
H), 7.02-7.12 (m, 1 H), 7.24 (ddd, J = 7.4, 4.8, 1.0 Hz, 1 H), 7.44
(s, 1 H), 7.73 (td, J = 7.7, 2.0 Hz, 1 H), 8.15 (s, 1 H), 8.23 (s,
1 H), 8.50-8.57 (m, 1 H) 25 ##STR00039## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.23 (t, J = 7.5 Hz, 3 H), 1.68 (d, J =
7.0 Hz, 3 H), 2.71 (q, J = 7.6 Hz, 2 H), 3.96 (s, 3 H), 5.71 (quin,
J = 7.2 Hz, 1 H), 6.05 (br. s., 2 H), 6.57-6.62 (m, 1 H), 6.83 (dd,
J = 8.5, 0.8 Hz, 1 H), 7.41 (t, J = 8.1 Hz, 1 H), 8.31 (d, J = 7.5
Hz, 1 H) 26 ##STR00040## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 1.05 (br. s., 3 H), 1.16 (br. s., 3 H), 1.60 (d, J =
7.0 Hz, 3 H), 3.20 (br. s., 2 H), 3.43 (br. s., 2 H), 5.60 (quin, J
= 7.3 Hz, 1 H), 6.11 (s, 2 H), 6.97 (dd, J = 8.3, 1.7 Hz, 1 H),
7.05 (d, J = 1.3 Hz, 1 H), 7.24 (ddd, J = 7.4, 4.8, 1.0 Hz, 1 H),
7.45 (d, J = 7.9 Hz, 1 H), 7.73 (td, J = 7.6, 1.9 Hz, 1 H), 8.15
(d, J = 7.9 Hz, 1 H), 8.24 (d, J = 8.4 Hz, 1 H), 8.50 - 8.56 (m, 1
H) 27 ##STR00041## .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.60 (d, J = 7.0 Hz, 3 H), 2.79 (d, J = 4.4 Hz, 3 H), 5.60 (quin, J
= 7.3 Hz, 1 H), 6.09 (s, 2 H), 7.24 (ddd, J = 7.4, 4.8, 1.0 Hz, 1
H), 7.41-7.48 (m, 2 H), 7.65 (d, J = 1.5 Hz, 1 H), 7.73 (td, J =
7.7, 2.0 Hz, 1 H), 8.15 (d, J = 7.9 Hz, 1 H), 8.24 (d, J = 8.6 Hz,
1 H), 8.48-8.56 (m, 2 H) 28 ##STR00042## .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 1.60 (d, J = 7.0 Hz, 3 H), 1.76-1.93 (m,
4 H), 3.37 (t, J = 6.5 Hz, 2 H), 3.47 (t, J = 6.8 Hz, 2 H), 5.60
(quin, J = 7.2 Hz, 1 H), 6.10 (s, 2 H), 7.12 (dd, J = 8.3, 1.7 Hz,
1 H), 7.22 (d, J = 1.5 Hz, 1 H), 7.23-7.26 (m, 1 H), 7.44 (d, J =
7.9 Hz, 1 H), 7.73 (td, J = 7.7, 1.8 Hz, 1 H), 8.15 (d, J = 7.9 Hz,
1 H), 8.23 (d, J = 8.6 Hz, 1 H), 8.50-8.56 (m, 1 H) 29 ##STR00043##
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 2.33- 2.42 (m, 3 H)
3.27 (s, 3 H) 3.64-3.80 (m, 2 H) 4.16-4.31 (m, 2 H) 4.69 (d, J =
5.50 Hz, 2 H) 6.12 (s, 2 H) 6.21-6.29 (m, 1 H) 6.59 (d, J = 7.48
Hz, 1 H) 6.82 (d, J = 7.70 Hz, 1 H) 7.37 (t, J = 8.25 Hz, 1 H) 8.37
(s, 1 H) 30 ##STR00044## .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 0.87 (t, J = 7.26 Hz, 3 H) 1.23-1.38 (m, 2 H) 1.38-1.49
(m, 2 H) 1.54 (d, J = 7.04 Hz, 3 H) 3.33-3.50 (m, 2 H) 5.38 (t, J =
7.26 Hz, 1 H) 6.10 (s, 2 H) 7.05 (dd, J = 7.04, 1.32 Hz, 1 H) 7.30
(dd, J = 8.47, 1.21 Hz, 1 H) 7.48 (dd, J = 8.36, 7.04 Hz, 1 H) 7.53
(dd, J = 1.87, 0.77 Hz, 1 H) 7.68 (t, J = 4.73 Hz, 1 H) 9.09 (d, J
= 1.98 Hz, 1 H) 9.39 (d, J = 8.14 Hz, 1 H) 31 ##STR00045## .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 2.28- 2.40 (m, 3 H) 3.90
(s, 3 H) 4.71 (d, J = 5.94 Hz, 2 H) 6.21 (d, J = 0.88 Hz, 1 H) 6.33
(br. s., 2 H) 6.46 (dd, J = 8.80, 3.52 Hz, 1 H) 7.26 (dd, J =
10.89, 8.69 Hz, 1 H) 8.49 (t, J = 5.72 Hz, 1 H) 32 ##STR00046## Not
available
SFC Purification Methods.
General Procedure
[0064] The Supercritical Fluid Chromatography (SFC) separation was
performed with supercritical CO.sub.2 and a modifier as specified
in the table using a column as specified in the table.
TABLE-US-00003 TABLE III Compounds of formula (I). The following
compounds were isolated SFC seperation. # Column Modifier 1
Chiralpak Diacel AS 20 .times. 250 mm iPrOH with 0.2% iPrNH2 2
Chiralpak Diacel AS 20 .times. 250 mm MeOH with 0.2% iPrNH2 3
Chiralpak Diacel AS 20 .times. 250 mm iPrOH with 0.2% iPrNH2 4
Chiralpak Diacel AD 30 .times. 250 mm iPrOH with 0.2% iPrNH2 5
Chiralpak Diacel AS 20 .times. 250 mm iPrOH with 0.4% iPrNH2 6
Chiralpak Diacel AS 20 .times. 250 mm EtOH with 0.2% iPrNH2 7
Chiralpak Diacel AS 20 .times. 250 mm iPrOH with 0.2% iPrNH2 8
Chiralpak Diacel AS 20 .times. 250 mm iPrOH with 0.2% iPrNH2 9
Chiralpak Diacel AD 30 .times. 250 mm EtOH with 0.2% iPrNH2 10
Chiralpak Diacel AS 20 .times. 250 mm EtOH with 0.4%iPrNH2 16
Chiralpak Diacel AD 30 .times. 250 mm EtOH with 0.2% iPrNH2 18
Chiralpak Diacel AS 20 .times. 250 mm iPrOH with 0.2% iPrNH2 20
Chiralpak Diacel AS 20 .times. 250 mm EtOH with 0.2% iPrNH2 21
Chiralpak Diacel AD 30 .times. 250 mm EtOH with 0.2% iPrNH2 25
Chiralpak Diacel AD 30 .times. 250 mm MeOH with 0.4% iPrNH2 26
Chiralpak Diacel AD 30 .times. 250 mm EtOH with 0.2% iPrNH2 27
Chiralpak Diacel AD 30 .times. 250 mm MeOH with 0.4% iPrNH2 28
Chiralpak Diacel AD 30 .times. 250 mm EtOH with 0.4% iPrNH2
[0065] For all compounds the first eluting compound was assigned as
*R.
[0066] *R means an enantiomeric pure configuration of which the
absolute stereochemistry is unknown.
Analytical Methods.
General Procedure
[0067] The High Performance Liquid Chromatography (HPLC)
measurement was performed using a LC pump, a diode-array (DAD) or a
UV detector and a column as specified in the respective methods. If
necessary, additional detectors were included (see table of methods
below).
[0068] Flow from the column was brought to the Mass Spectrometer
(MS) which was configured with an atmospheric pressure ion source.
It is within the knowledge of the skilled person to set the tune
parameters (e.g. scanning range, dwell time . . . ) in order to
obtain ions allowing the identification of the compound's nominal
monoisotopic molecular weight (MW). Data acquisition was performed
with appropriate software.
[0069] Compounds are described by their experimental retention
times (Rt) and ions. If not specified differently in the table of
data, the reported molecular ion corresponds to the [M+H].sup.+
(protonated molecule) and/or [M-H].sup.- (deprotonated molecule).
In case the compound was not directly ionizable the type of adduct
is specified (i.e. [M+NH.sub.4].sup.+, [M+HCOO].sup.-, etc. . . .
). For molecules with multiple isotopic patterns (Br, Cl . . . ),
the reported value is the one obtained for the lowest isotope mass.
All results were obtained with experimental uncertainties that are
commonly associated with the method used.
[0070] Hereinafter, "SQD" means Single Quadrupole Detector, "MSD"
Mass Selective Detector, "RT" room temperature, "BEH" bridged
ethylsiloxane/silica hybrid, "DAD" Diode Array Detector, "HSS" High
Strength silica.
TABLE-US-00004 TABLE IV LCMS Method codes (Flow expressed in
mL/min; column temperature (T) in .degree. C., Run time in
minutes). Flow Method Mobile Column Run code Instrument Column
phase gradient T time B7010 Waters: Waters: A: 95% From 100% A to
0.8 3.5 B7014 Acquity .RTM. HSS T3 CH.sub.3COONH.sub.4 5% A in 2.10
min, 55 UPLC .RTM.- (1.8 .mu.m, 10 mM + 5% to 0% A in DAD and
2.1*100 mm) CH.sub.3CN 0.90 min, to 5% A SQD B: CH.sub.3CN in 0.5
min B8011 Waters: Waters: A: 95% From 95% A to 0.8 2 B8002 Acquity
.RTM. BEH C18 CH.sub.3COONH.sub.4 5% A in 1.3 min, 55 UPLC .RTM.-
(1.7 .mu.m, 10 mM + 5% held for 0.7 min. DAD and 2.1*50 mm)
CH.sub.3CN SQD B: CH.sub.3CN B9007 Waters: Waters: A: 95% From 100%
A to 0.8 3.5 B9008 Acquity .RTM. HSS T3 CH.sub.3COONH.sub.4 5% A in
2.10 min, 55 UPLC .RTM.- (1.8 .mu.m, 10 mM + 5% to 0% A in DAD and
2.1*100 mm) CN.sub.3CH 0.90 min, to 5% A SQD B: CH.sub.3CN in 0.5
min
TABLE-US-00005 TABLE V Compounds of formula (I). The following
compounds were characterized according to one of the methods
described above. Mass Method Retention Found # code Time (min) (M +
H) 1 B701067014 0.61 266 2 B701067014 1.59 296 3 B9007B9008 1.61
350 4 B8011B8002 0.69 284 5 B701067014 1.39 280 6 B701067014 1.31
296 7 B8011B8002 0.78 300 8 B9007B9008 1.32 324 9 B9007B9008 1.29
284 10 B9007B9008 1.31 302 11 B8011B8002 0.69 274 12 B701067014
1.26 286 13 B8011B8002 0.64 274 14 B701067014 1.32 274 15
B8011B8002 0.77 325 16 B8011B8002 0.62 299 17 B8011138002 0.53 311
18 B8011138002 0.79 284 19 B8011138002 0.65 270 20 B8011138002 0.84
310 21 B8011138002 0.61 314 22 B8011138002 0.56 323 23 B8011138002
0.59 337 24 B8011138002 0.70 365 25 B8011138002 0.75 315 26
B8011138002 0.69 365 27 B8011138002 0.56 323 28 B8011138002 0.66
363 29 B8011138002 0.70 330 30 B9007139008 1.04 286 31 B9007139008
1.36 304 32 B9007139008 1.46 348
Biological Activity of Compounds of Formula (I)
Description of Biological Assays
Assessment of TLR7 and TLR8 Activity
[0071] The ability of compounds to activate human TLR7 and/or TLR8
was assessed in a cellular reporter assay using HEK293 cells
transiently transfected with a TLR7 or TLR8 expression vector and
NF.kappa.B-luc reporter construct.
[0072] Briefly, HEK293 cells were grown in culture medium (DMEM
supplemented with 10% FCS and 2 mM Glutamine). For transfection of
cells in 15 cm dishes, cells were detached with Trypsin-EDTA,
transfected with a mix of CMV-TLR7 or TLR8 plasmid (1700 ng),
NF.kappa.B-luc plasmid (850 ng) and a transfection reagent and
incubated for 48 h at 37.degree. C. in a humidified 5% CO.sub.2
atmosphere. Transfected cells were then washed in PBS, detached
with Trypsin-EDTA and resuspended in medium to a density of
1.25.times.10.sup.5 cells/mL. Forty microliters of cells were then
dispensed into each well in 384-well plates, where 200 nL of
compound in 100% DMSO was already present. Following 6 hours
incubation at 37.degree. C., 5% CO.sub.2, the luciferase activity
was determined by adding 15 .mu.L of Steady Lite Plus substrate
(Perkin Elmer) to each well and readout performed on a ViewLux
ultraHTS microplate imager (Perkin Elmer). Dose response curves
were generated from measurements performed in quadruplicates.
Lowest effective concentrations (LEC) values, defined as the
concentration that induces an effect which is at least two fold
above the standard deviation of the assay, were determined for each
compound.
[0073] Compound toxicity was determined in parallel using a similar
dilution series of compound with 40 .mu.L per well of cells
transfected with the CMV-TLR7 construct alone (1.25.times.10.sup.5
cells/mL), in 384-well plates. Cell viability was measured after 6
hours incubation at 37.degree. C., 5% CO.sub.2 by adding 15 .mu.L
of ATP lite (Perkin Elmer) per well and reading on a ViewLux
ultraHTS microplate imager (Perkin Elmer). Data was reported as
CC.sub.50.
[0074] In parallel, a similar dilution series of compound was used
(200 nL of compound in 100% DMSO) with 40 .mu.L per well of cells
transfected with NF.kappa.B-luc reporter construct alone
(1.25.times.10.sup.5 cells/mL). Six hours after incubation at
37.degree. C., 5% CO.sub.2, the luciferase activity was determined
by adding 15 .mu.l of Steady Lite Plus substrate (Perkin Elmer) to
each well and readout performed on a ViewLux ultraHTS microplate
imager (Perkin Elmer). Counterscreen data is reported as LEC.
Activation of ISRE Promoter Elements
[0075] The potential of compounds to induce IFN-I was also
evaluated by measuring the activation of interferon-stimulated
responsive elements (ISRE) by conditioned media from PBMC. The ISRE
element of sequence GAAACTGAAACT (SEQ ID: 1) is highly responsive
to the STAT1-STAT2-IRF9 transcription factor, activated upon
binding of IFN-I to their receptor IFNAR (Clontech, PT3372-5W). The
plasmid pISRE-Luc from Clontech (ref. 631913) contains 5 copies of
this ISRE element, followed by the firefly luciferase ORF. A HEK293
cell line stably transfected with pISRE-Luc (HEK-ISREluc) was
established to profile the conditioned PBMC cell culture media.
[0076] Briefly, PBMCs were prepared from buffy coats of at least
two donors using a standard Ficoll centrifugation protocol.
Isolated PBMCs were resuspended in RPMI medium supplemented with
10% human AB serum and 2.times.10.sup.5 cells/well were dispensed
into 384-well plates containing compounds (70 .mu.L total volume).
After overnight incubation, 10 .mu.L of supernatant was transferred
to 384-well plates containing 5.times.10.sup.3 HEK-ISREluc
cells/well in 30 .mu.L (plated the day before). Following 24 hours
of incubation, activation of the ISRE elements was measured by
assaying luciferase activity using 40 .mu.L/well Steady Lite Plus
substrate (Perkin Elmer) and measured with ViewLux ultraHTS
microplate imager (Perkin Elmer). The stimulating activity of each
compound on the HEK-ISREluc cells was reported as LEC value,
defined as the compound concentration applied to the PBMCs
resulting in a luciferase activity at least two fold above the
standard deviation of the assay. The LEC in turn indicates the
degree of ISRE activation on transfer of a defined amount of PBMC
culture medium. Recombinant interferon .alpha.-2a (Roferon-A) was
used as a standard control compound.
TABLE-US-00006 TABLE VI BIOLOGICAL ACTIVITY. Human TLR 7 Human TLR
8 HEK-ISRE luc # (LEC) .mu.M (LEC) .mu.M (LEC) .mu.M 1 0.72 >25
0.61 2 0.94 >25 0.49 3 0.76 >25 0.47 4 0.92 19.8 0.59 5 0.53
14.7 0.11 6 3.75 >25 0.64 7 0.82 16.4 0.38 8 4.94 NA 2.11 9 5.21
>25 1.68 10 0.42 12.3 0.11 11 0.45 3.09 0.082 12 0.047 1.94
0.036 13 0.46 5.22 0.12 14 0.65 >25 0.13 15 0.61 >25 0.56 16
2.44 9.14 0.55 17 0.83 5.51 0.16 18 8.25 24.3 7.83 19 0.11 1.74
0.051 20 1.46 >25 0.62 21 6.1 8.85 0.54 22 14.7 >25 2.20 23
6.67 >25 1.62 24 14.3 11.0 1.75 25 1.95 6.62 0.49 26 2.14 >25
7.33 27 8.24 >25 5.04 28 2.24 >25 1.57 29 0.082 8.15 NA 30
0.63 9.0 0.14 31 0.74 >25 0.46 32 NA NA NA NA = not available.
All compounds showed no toxicity up to the highest tested
concentration. All compounds showed no activity (LEC >25 .mu.M)
in the HEK 293 NF-kB counterscreen assay described above.
Sequence CWU 1
1
1112DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gaaactgaaa ct 12
* * * * *